In the field of current biological detection, microspheres have been widely used as a solid carrier in vitro diagnosis to identify, capture, control and transport target molecules to be tested. On this basis, a further goal of research and experiment is to achieve multiplex detection, i.e. various target molecules to be tested can be detected simultaneously in one detection, and to add detection items in multiplex detection. Therefore, microspheres carrying different specific ligands should be marked, i.e. the microspheres should be encoded so that they can be identified according to the marker thereon during the detection.
An early encoding mode is adding an organic fluorescent dye in microspheres. The fluorescent dye will have specific fluorescence emission spectrum, including intensity and emission wavelength of the fluorescence, when excited by a laser at a specific wavelength. When different organic fluorescent dyes are used, different fluorescence emission spectrums can be obtained after excitation, thereby identifying the microspheres. However, following disadvantages exist when organic fluorescent dyes are used.
(1) It is difficult to select an organic dye, because the Stocks shift of the organic dye itself is relatively small and every dye has its best excitation wavelength, in order to reach their optimum fluorescence emission intensity, usually it is needed to use lasers with different excitation wavelengths, which makes that a testing equipment must be equipped with multiple lasers and it is difficult to achieve miniaturization of the detection equipment.
(2) The stability of organic dyes is relatively poor, and it is prone to photobleaching or photoquenching. Once above phenomena occurs, the fluorescence intensity or the fluorescence emission wavelength will change, thus affecting the encoding precision and accuracy degree.
(3) The emission spectrum of organic dyes is wide and asymmetrical. When two kinds of organic dyes having different emission spectrum are hybrid encoded, the fluorescence emission spectrum are easily to be superimposed on each other, which makes that the encoding ability is low and they are easy to interfere with each other.
Quantum dot, also known as semiconductor nanocrystal, is stable nanocrystal particle with a size of 1-100 nm consisted of elements selected from group IIB/VIA (such as CdSe, etc.) or group IIIA/VA (such as InP, GaAs, etc.). It can accept laser excitation and produce fluorescence. The special structure makes it have unique optical properties which are unmatched for ordinary fluorescence material. There has achieved some breakthrough progress in its application in biological sciences, and related technologies by using quantum dots instead of organic fluorescent dyes to encode microspheres have been developed. Compared with organic fluorescent dyes, quantum dot has following advantages:
(1) The quantum dot has very wide Stocks displacement, and emission spectrum of quantum dots with different particle size is different from each other. The larger the particle size of the quantum dot is, the greater its emission wavelength is. Therefore, by regulating the particle size of the quantum dot, spectrum with different fluorescence colors can be emitted when quantum dots with different particle sizes are excited by using same laser.
(2) The quantum dot has high and stable fluorescence quantum yield, and photobleaching or photo-quenching effect is relatively weak, so the prepared encoded microsphere is less prone to cross color or garbled code.
(3) The emission spectrum of the quantum dot is narrow and symmetrical, compared with organic fluorescent dyes, its encoding ability is stronger and less prone to be interfered.
Although the microspheres encoded with quantum dots have provided specific ligand carrier which is easier to identify for multiplex detection, due to the inherent differences among various detection items in multiplex detection, it is difficult to make all of detection items in their optimal reaction conditions during multiplex detection, which inevitably cause differences in sensitivity among different detection items. Meanwhile, the non-specific adsorption of each probe molecule will also cause a certain degree of detection error.
The latest study has found that, compared with single microspheres having smooth surface, assembled microspheres having topology formed by connecting nano-sized microspheres to the surface of micron-sized or submicron-sized microspheres, as a solid carrier in biological detection, can significantly improve the detection sensitivity and reduce non-specific adsorption. Therefore, the assembled microspheres are more suitable for multiplex detection. Related arts of assembled microspheres may refer to Chinese Application CN103134926A.
In the field of biology, both quantum dot encoding and assembled microspheres are all advanced technology, and currently there is no related technology on assembled microspheres encoded with quantum dots formed by combining the above two and preparation thereof. Meanwhile, it can be forecasted that, on the premise that the types of quantum dots that can be employed are certain, compared with the single microspheres, encoding assembled microspheres can generate more encoding amount or types of identifiable carrier, which also provides necessary conditions for adding detection items in multiplex detection.
Therefore, the skilled in the art devote themselves to develop an assembled microsphere encoded with quantum dots which is more suitable for multiplex detection in bioassay and preparation method thereof.